Project Summary The predoctoral phase of this proposal will focus on the biochemical and biophysical studies of the enzyme telomerase. Telomerase is a ribonucleoprotein that maintains telomere lengths in rapidly dividing cells, thereby counteracting the chromosome shortening that is inherent to each round of cell division. Without telomerase, cells undergo senescence when telomere lengths become critically short. Telomerase is upregulated in about 90% of cancers due to the need for telomere maintenance in rapidly dividing cells making it an attractive drug target. Cancer therapeutics that target active telomerase directly have remained elusive however. The structure and function of human telomerase is still poorly understood, and any new insight would greatly benefit the development of novel cancer therapeutics. The dissertation project focuses on using single molecule techniques to study the conformation and dynamics of human telomerase. The minimal human telomerase is composed of two subunits, a protein component (TERT) that contains four evolutionarily conserved domains and an RNA component (TER) which contains the integral non-coding RNA template from which telomeres are reverse transcribed. Telomerase is unique in its ability to reverse transcribe multiple telomere repeats during a single DNA binding event, but the details of how telomerase maintain this processive action remains poorly understood. Additionally, details regarding how TERT and TER interact during catalysis, and how individual TERT domains regulate telomerase dynamics, remain unknown. Specifically, knowledge about the function and dynamics of two TERT domains, the Telomerase Essential N-terminal (TEN) domain and the C-terminal extension (CTE) remain especially poorly understood. This project will test the hypothesis that the TEN and CTE domains drive the processive action of telomerase via coordinated dynamic rearrangement of the telomere/template hybrid into its initial bound state at the end of each telomere repeat addition event. Single molecule Frster Resonance Energy Transfer (smFRET), in combination with a novel protein labeling scheme, will be used to probe the conformation and dynamics of these domains during different functional states. In addition, computational modeling in collaboration with the Das lab at Stanford will enable the construction of a refined working model of human telomerase during catalysis. Completion of this proposal will aid in the development of telomerase targeted cancer therapeutics. In addition, this proposal will facilitate the smooth transition from studying RNP structure and function during the predoctoral phase, to the study of long non-coding RNA (lncRNA) structure and its roles in the development of cancer during the postdoctoral phase. The techniques and approaches learned during the dissertation research will lend themselves perfectly to the study of RNA structure, which to date has remained a difficult area to study. The timing of the proposed postdoctoral work focused on lncRNA structure and cancer will be ideal considering the current stage of lncRNA biology and overall, this proposal is well suited to provide valuable insight into how RNA structure contributes to cancer development. !